Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, oomycetes and nematodes. Plants recognize and resist many invading phytopathogens by inducing a rapid defense response. Recognition is often due to the interaction between a dominant or semi-dominant resistance (R) gene product in the plant and a corresponding dominant avirulence (Avr) gene product expressed by the invading phytopathogen. R-gene triggered resistance often results in a programmed cell-death, that has been termed the hypersensitive response (HR). The HR is believed to constrain spread of the pathogen.
How R gene products mediate perception of the corresponding Avr proteins is mostly unclear. It has been proposed that phytopathogen Avr products function as ligands, and that plant R products function as receptors. In this receptor-ligand model binding of the Avr product to a corresponding R product in the plant initiates the chain of events within the plant that produces HR leads to disease resistance. In an alternate model the R protein perceives the action rather than the structure of the Avr protein. In this model the Avr protein is believed to modify a plant target protein (pathogenicity target) in order to promote pathogen virulence. The modification of the pathogenicity protein is detected by the matching R protein and triggers a defense response. Experimental evidence suggests that some R proteins act as Avr receptors while others detect the activity of the Avr protein.
The production of transgenic plants carrying a heterologous gene sequence is now routinely practiced by plant molecular biologists. Methods for incorporating an isolated gene sequence into an expression cassette, producing plant transformation vectors, and transforming many types of plants are well known. Examples of the production of transgenic plants having modified characteristics as a result of the introduction of a heterologous transgene include: U.S. Pat. No. 5,719,046 to Guerineau (production of herbicide resistant plants by introduction of bacterial dihydropteroate synthase gene); U.S. Pat. No. 5,231,020 to Jorgensen (modification of flavenoids in plants); U.S. Pat. No. 5,583,021 to Dougherty (production of virus resistant plants); and U.S. Pat. No. 5,767,372 to De Greve and U.S. Pat. No. 5,500,365 to Fischoff (production of insect resistant plants by introducing Bacillus thuringiensis genes).
In conjunction with such techniques, the isolation of plant R genes has similarly permitted the production of plants having enhanced resistance to certain pathogens. Since the cloning of the first R gene, Pto from tomato, which confers resistance to Pseudomonas syringae pv. tomato (Martin et al. (1993) Science 262: 1432-1436), a number of other R genes have been reported (Hammond-Kosack & Jones (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:575-607). A number of these genes have been used to introduce the encoded resistance characteristic into plant lines that were previously susceptible to the corresponding pathogen. For example, U.S. Pat. No. 5,571,706 describes the introduction of the Ngene into tobacco lines that are susceptible to Tobacco Mosaic Virus (TMV) in order to produce TMV-resistant tobacco plants. WO 95/28423 describes the creation of transgenic plants carrying the Rps2 gene from Arabidopsis thaliana, as a means of creating resistance to bacterial pathogens including Pseudomonas syringae, and WO 98/02545 describes the introduction of the Prf gene into plants to obtain broad-spectrum pathogen resistance.
Bacterial spot disease of tomato and pepper, caused by the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), can be devastating to commercial production of these crops in areas of the world with high humidity and heavy rainfall. While control of Xcv in commercial agriculture is based largely on the application of pesticides, genetic resistance to bacterial spot disease has been described in both tomato and pepper (Cook and Stall (1963) Phytopathology 53: 1060-1062; Cook and Guevara (1984) Plant Dis. 68: 329-330; Kim and Hartman (1985) Plant Dis. 69: 233-235; Jones and Scott (1986) Plant Dis. 70: 337-339). Of the two hosts, genetic resistance in pepper has been better characterized. Several single loci (Bs), Bs2, and Bs3) that confer resistance in a “gene-for-gene” manner have been identified (Hibberd et al. (1987) Phytopathology 77: 1304-1307). Moreover, the corresponding avirulence genes (avrBs1, avrBs2, and avrBs3) have been cloned from Xcv (Swanson et al. (1988) Mol Plant-Microbe Interact. 1:5-9; Minsavage et al. (1990) Mol. Plant-Microbe Interact. 3: 41-47). Genetic and molecular characterization of these avirulence genes has provided a great deal of information concerning the interaction between Xcv and pepper (Kearney et al. (1988) Nature 332: 541-543; Kearney and Staskawicz (1990) Nature 346: 385-386; Herbers et al. (1992) Nature 356: 172-174; Van der Ackerveken et al. (1992) Plant J. 2: 359-366). More recently, the Bs3 gene of pepper has been isolated and sequenced (U.S. Pat. No. 6,262,343)
Xcv employs a type III secretion (T3S) system to inject an arsenal of about 20 effector proteins into the host cytoplasm that collectively promote virulence (Thieme et al. (2005) J. Bacteriol. 187:7254). R protein mediated defense in response to Xcv effector proteins is typically accompanied by a programmed cell death response referred to as the hypersensitive response (HR). AvrBs3 is one Avr protein that R proteins recognize and is a member of a Xanthomonas family of highly conserved proteins (Schornack et al. (2006) J. Plant Physiol. 163:256). The central region of AvrBs3 consists of 17.5 tandem near-perfect 34-amino-acid (aa) repeat units that determine avirulence specificity (Herbers et al. (1992) Nature 356:172). AvrBs3 contains also nuclear localization signals (NLSs) and an acidic transcriptional activation domain (AD) (Szurek et al. (2001) Plant J. 26:523; Szurek et al. (2002) Mol. Microbiol. 46:13), similar to eukaryotic transcription factors, and induces host gene transcription (Marois et al. (2002) Mol Plant-Microbe Interact. 15:637-646). Mutations in the NLS or AD of AvrBs3 abolish pathogen recognition by the matching pepper R gene Bs3 (Szurek et al. (2001) Plant J. 26:523; Van den Ackerveken et al. (1996) Cell 87:1307) suggesting that recognition involves transcriptional activation of host genes.
The isolation to the Bs3 gene from pepper would provide researchers with the opportunity to further study this recognition process while providing an R gene that can be used to produce transgenic plants having increased resistance to phytopathogens.